幽门螺杆菌东、西方株CagA的序列差异及其对胃癌细胞生长与凋亡的影响

sequences difference of CagA between East Asian strain and Western strain of Helicobacter pylori and its impact on the proliferation and apoptosis of gastric cancer cells

摘要:

【背景】幽门螺杆菌(Helicobacter pylori，H. pylori)是胃癌的主要致病因素，其分泌的细胞毒素相关基因A蛋白(Cytotoxin associated gene A，CagA)是目前已知唯一能被H. pylori注入胃上皮细胞并模拟细胞内蛋白发挥作用的癌蛋白，参与胃癌的发生发展。【目的】比较H. pylori东亚株和西方株CagA结构差异，初步探讨H. pylori-CagA对胃癌细胞增殖与凋亡的影响。【方法】对H. pylori东亚株和西方株CagA的核酸及氨基酸序列进行生物信息学分析，构建含东亚株和西方株cagA基因的真核表达载体，转染胃癌细胞AGS，用Western blot法检测CagA蛋白的表达，用CCK8法测定细胞的生长曲线，流式细胞术检测细胞凋亡。【结果】生物信息学分析发现H. pylori东亚、西方菌株CagA的核酸序列和氨基酸序列均存在特征性差异。构建了含东亚、西方菌株cagA基因的表达载体[命名为GZ7/cagA (东亚株)和26695/cagA (西方株)]。与空载体组比较，GZ7/cagA和26695/cagA转染组均表达CagA蛋白，两组比较表达量无显著性差异，GZ7/cagA转染组细胞生长显著增加，而26695/cagA转染组细胞生长显著降低(P<0.05)。GZ7/cagA转染组、26695/cagA转染组细胞的凋亡率分别为7.23±0.96及9.17±1.40，均高于空载体组(5.03±0.63)，差异有统计学意义(P<0.05)。【结论】东亚株与西方株CagA之间有结构和功能的差异，东亚株CagA能促进细胞增殖，而西方株CagA却抑制细胞增殖，但两者均能促进细胞凋亡。

Abstract:

[Background] Helicobacter pylori (H. pylori) has been described as a main pathogenic factor for gastric cancer. Protein product of the cytotoxin associated gene A (CagA) has been known as the only oncoprotein that is secreted and injected into gastric epithelial cells by the bacteria, thus mediating the development of gastric cancer. [Objective] To compare the sequence difference of CagA between the East Asian strain and Western strain of H. pylori, and explore the effects of H. pylori-CagA on the proliferation and apoptosis of gastric cancer cells. [Methods] DNA and amino acid sequences of CagA in East Asian strain and Western strain were analyzed. A eukaryotic expression vector containing the cagA gene from each strain was constructed and transferred into AGS gastric cancer cells. The CagA protein was assessed using Western blot. Cell growth curve and apoptosis were determined using CCK8 and flow cytometry, respectively. [Results] Comparison of the DNA and amino acid sequences in the CagA between the two strains revealed some characteristic differences. The expression vectors of H. pylori-cagA gene were constructed successfully and named as GZ7/cagA (East Asian strain) and 26695/cagA (Western strain). Compared with cells transfected with the empty vector, the cells transfected with the East Asian strain and the Western strain derived cagA expressed CagA proteins, but there is no difference in protein levels between the two groups. The cells transfected with the East Asian strain and the Western strain derived cagA grew significantly fast or slower, respectively (P<0.05). The apoptotic rates of cells expressing the East Asian strain derived CagA and the Western strain derived CagA were 7.23±0.96 and 9.17±1.40, respectively, both being higher than that of the cells expressing the empty vector (5.03±0.63) (P<0.05). [Conclusion] There are structural and functional differences in the CagA between the East Asian and Western strains of H. pylori. While the one in the East Asian strain promotes the proliferation, Western CagA inhibits the proliferation of gastric cancer cells. However, the CagA in the both strains promotes apoptosis of the cells.